1. Field of the Invention
The present invention relates to a spectroscopy analysis apparatus using a fluorescence correlation spectroscopy analysis technique for analyzing a state of fluorescence molecules by analyzing fluctuation of the fluorescence molecules in a biological sample. In particular, the present invention relates to a spectroscopy analysis apparatus for analyzing a correlation of fluorescence intensity between different measuring points.
2. Description of the Related Art
A fluorescence correlation spectroscopy analysis technique (FCS technique) is a technique of analyzing fluctuation of light produced by a Brown motion of fluorescence molecules in a microscopic monitoring region in a field of view of a microscope to obtain an auto-correlation function of fluorescence intensity and analyzing a dispersion time or an average number of molecules on a molecule by molecule basis. This technique is described in detail in “single molecule detection using a fluorescence correlation spectroscopy technique”, Authored by KINJO, “Protein nuclear acid enzyme”, 1999, vol. 44NO9 1431-1438, for example. When the fluorescence intensity is defined as I(t), the auto-correlation function can be expressed in the form of formula (1).
                              g          ⁡                      (            τ            )                          =                                            1              T                        ⁢                                          ∫                0                T                            ⁢                                                l                  ⁡                                      (                    t                    )                                                  ⁢                                  l                  ⁡                                      (                                          t                      +                      τ                                        )                                                  ⁢                                                                  ⁢                                  ⅆ                  t                                                                                        [                                                1                  T                                ⁢                                                      ∫                    0                    T                                    ⁢                                                            l                      ⁡                                              (                        t                        )                                                              ⁢                                                                                  ⁢                                          ⅆ                      t                                                                                  ]                        2                                              (        1        )            
FIG. 1 is a diagram showing an example of an optical system for used in measurement by such an FCS technique. In FIG. 1, a laser light source 100 is used as an excitation light source. The laser beam from the laser light source 100 is reflected on a dichroic mirror 101, and then is incident to an objective lens 102. A sample 103 labeled with a fluorescence dye is placed at a focal point of the objective lens 102. The laser beam focused at the focal portion by the objective lens 102 excites the fluorescence dye and induces fluorescence. The fluorescence emitted from the fluorescence dye of the sample 103 is captured again by the objective lens 102, and then reaches the dichroic mirror 101. The dichroic mirror 101 has optical characteristics that reflect excited light and transmits fluorescence. Thus, the fluorescence from the sample 103 passes through the dichroic mirror 101, and is focused by a focusing lens 104. A pin hole 105 is disposed at the focal point of the focusing lens 104. The fluorescence from a position other than the focal point of the objective lens 102 is interrupted by the pin hole 105, whereby high spatial resolution can be obtained. The fluorescence having passed through the pin hole 105 is incident to a photodetector 106, and the fluctuation of the fluorescence intensity is measured.
FIG. 2 is a diagram showing an example of an optical system such that the fluctuations of the fluorescence intensities at two measurement points can be measured at the same time. In FIG. 2, a laser light source 200 is used as an excitation light source. The laser beam which is the excited light from the laser light source 200 is split into two luminous fluxes by a beam splitter 201. The laser beams split into two luminous fluxes are reflected on mirrors 202 and 203, respectively, and combined again by a beam splitter 204. The two laser beams combined by the beam splitter 204 are reflected on the mirrors 202 and 203 such that their light axes are slightly shifted from each other. The combined light fluxes enter an objective lens 206 after they have been reflected on the dichroic mirror 205, and connects their focal points at two points which are slightly spaced from each other on a sample 207. Then, the fluorescence emitted from a focal region of each of the two points on the sample 207 is captured again by the objective lens 206. The captured fluorescence passes through the dichroic mirror 205 and is focused by a focusing lens 208 to connect their focal points at two points which correspond to the focal regions of the respective two points. Then, from this focal point, the resulting lights are incident to photodetectors 210a and 210b through optical fibers 209a and 209b, respectively, and the fluctuation of the fluorescence intensity of each light is measured. In this example, although no pin hole is used, core diameters of the optical fibers 209a and 209b function as pin holes.
A fluorescence cross-correlation spectroscopy analysis technique (FCCS technique) is devised as an analysis technique having enhanced the above-described fluorescence correlation spectroscopy analysis technique (FCS technique). The fluorescence cross-correlation spectroscopy analysis technique (FCCS technique) is a technique of obtaining a cross-correlation function between different fluorescence signals to analyze a correlation therebetween. In the fluorescence cross-correlation spectroscopy analysis technique (FCCS technique), there is a case in which a correlation is obtained with respect to two fluorescence dyes in an identical measurement point or a case in which a correlation is obtained between two measurement points. These cross-correlation functions are expressed in the form of formula (2). In formula (2), IA(t) and IB(t) designates their respective fluorescence intensity signals.
                              g          ⁡                      (            τ            )                          =                                            1              T                        ⁢                                          ∫                0                T                            ⁢                                                                    l                    A                                    ⁡                                      (                    t                    )                                                  ⁢                                                      l                    B                                    ⁡                                      (                                          t                      +                      τ                                        )                                                  ⁢                                                                  ⁢                                  ⅆ                  t                                                                                        1              T                        ⁢                                          ∫                0                T                            ⁢                                                                    l                    A                                    ⁡                                      (                    t                    )                                                  ⁢                                                                  ⁢                                                      ⅆ                    t                                    ·                                      1                    T                                                  ⁢                                                      ∫                    0                    T                                    ⁢                                                                                    l                        B                                            ⁡                                              (                        t                        )                                                              ⁢                                                                                  ⁢                                          ⅆ                      t                                                                                                                              (        2        )            
In the identical measurement point, cross-correlation analysis between dual-color fluorescence dyes is described in detail in “Dual-Color Fluorescence Cross-Correlation Spectroscopy for Multicomponent Diffusional Analysis in Solution, P. Schwille et al, Biophysical Journal 1997, 72, 1878-1886. This analysis is used for analysis of interaction between the molecules labeled with dual-color fluorescence dyes. A method of analyzing a correlation between different points is described in “Two-Beam Cross-Correlation: A method To Characterize Transport Phenomena in Micrometer Sized Structures”, M. Brinkmeier et al, Anal. Chem. 1999, 71, 609-616, and a method of measuring a velocity and a direction of a flow of a fluid is introduced.
The fluorescence correlation spectroscopy analysis technique (FCS technique) and the fluorescence cross-correlation spectroscopy analysis technique (FCCS technique) are noticeable in advantage that measurement can be invasively carried out. In recent years, these techniques have been used for a non-homogenous sample such as a cell system. While avoiding the vicinity of a critical surface of a container in which a specific phenomenon such as adsorption is likely to occur, all locations are basically uniform in a solution system, and thus, it is possible to grasp an outlook of an entire system by measuring one point in solution. However, because different events occur at individual locations in a non-homogenous system such as a cell system, a measurement result greatly depends on a measurement location. In addition, in a non-homogenous system, in particular, in a cell system, the events between different points are associated with each other. There are many cases in which a certain event at a certain point has a temporal and spatial association such that it causes another event at another point. If an attempt is made to properly comprehend such a system, it is indispensable to carry out measurements between the different points at the same time. In recent years, there has been a growing demand for such a measurement among researchers.
As an optical system for carrying out fluorescence measurement among a plurality of measurement points at the same time, there has been proposed a method using a plurality of excitation light sources and a plurality of detectors, as disclosed in U.S. Pat. No. 6,320,196.
As another method, there has been proposed a method using an optical system which splits light excited from one excitation light source by a beam splitter and supplies the split light beams excited from such one excitation light source to a plurality of measurement points, as disclosed in Jpn. Pat. Appln. KOKAI Publication No. 9-113448.
In the method disclosed in U.S. Pat. No. 6,320,196, however, equipment becomes complicated because a plurality of optical systems each having an excitation light source and a detector are disposed. Further, a distance between measurement points is limited by a gap between the detectors, so that it is difficult to measure very close measurement points while individually tracking them.
In the method disclosed in Jpn. Pat. Appln. KOKAI Publication No. 9-113448, detectors are used independently on a measurement point by point basis, and thus, a complicated detecting optical system is unavoidable.
An optical system for use in fluorescence correlation spectroscopy analysis is used in combination of a laser and a photodetector with ultra-high sensitivity enabling single photon measurement. In general, these optical parts are very expensive. For this reason, installing these measuring systems in plurality makes equipment very expensive and large-sized. Moreover, in the case where a plurality of measurement points are set in a single cell, a gap between the measurement points becomes very small, is not always constant, and depends on cells targeted for measurement. In the conventional methods, it has been difficult to cope with these requests.